Compounds and Compositions That Bind and Stabilize Transthyretin and Their Use for Inhibiting Transthyretin Amyloidosis and Protein-Protein Interactions

ABSTRACT

Disclosed herein are compounds and compositions thereof which find use in increasing stability of proteins particularly proteins that tend to misfold and form aggregates. Also provided herein are methods for using these compounds and compositions for increasing stability of proteins and thereby decreasing aggregate formation by these proteins. Also disclosed herein are heterobifunctional compounds that include a TTR binding compound connected to a targeting moiety via a linker, for use in disrupting PPIs of a target protein.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/745,089, filed Dec. 21, 2012, which application isincorporated herein by reference in its entirety.

INTRODUCTION

Protein aggregation underlies a large number of human disorders,including some of the most common diseases observed in the agingpopulation, including systemic and CNS amyloidoses (Selkoe et al. NatCell Biol 6:1054-1061 (2004); Falk et al., N. Engl. J. Med. 337:898-909(1997)). Recently this process has also been implicated as an importantmechanism in cellular senescence (Haigis et al., Mol Cell 40:333-344(2010)). Aggregation of disease-associated peptides or proteins canoccur in different sub-cellular compartments and either affect specifictissues or spread systemically (Stefani et al., Biochimica et biophysicaacta 1739:5-25 (2004)). Data from biophysical, cellular and animalmodels indicate that a number of genetic and environmental factorscontribute to in vivo protein misfolding, aggregation and amyloid fibrilformation (amyloidogenesis). Protein misfolding and amyloid formation isbelieved to be intimately involved in the pathogenic mechanisms of humanamyloid diseases based on the demonstrated cytotoxicity of in vitroaggregated proteins/peptides. A number of observations in patients alsoindicate that amyloid formation is intimately linked to diseasepathogenesis. Such observations include: lower levels of amyloidobserved in the CNS of age-matched controls relative to Alzheimerdisease patients and the correlation of improved health with theclearance of amyloid in Familial Amyloid polyneuropathy (FAP) patients,following liver transplantation to replace mutant TTR with wild-typeTTR. Hence, there is strong interest in identifying ways to prevent theconformational changes that result in the formation of protein/peptideaggregates and amyloid formation.

Transthyretin (TTR or prealbumin) is one of more than 30 proteins whoseaggregation can cause disease (Selkoe et al., Nature 426:900-904 (2003);Reixach et al., Proc. Natl. Acad. Sci. USA 101:2817-2822 (2004)). TTR isa 127-amino-acid, s-sheet-rich protein, which forms homotetramers, andis primarily synthesized by the liver and secreted into the blood. Usingorthogonal binding surfaces TTR binds to both thyroxine (T₄) and holoretinol-binding protein (RBP). TTR is the main carrier of RBP, but dueto the presence of two other T₄ transport proteins it is only a back-upcarrier for T₄ in humans (<1% T₄ bound). The two T₄-binding pockets,which remain largely unoccupied in humans, are formed by the weakerdimer-dimer interface (Connelly et al., Curr Opin Struct Biol 20:54-62(2010)). Each of the two T₄ binding sites created by the dimer-dimerinterface of the TTR homotetramer consists of an outer binding subsite,an inner binding subsite, and an intervening interface that are composedof pairs of symmetric hydrophobic depressions referred to as halogenbinding pockets (HBPs), in which the iodine atoms of T₄ reside (Wojtczaket al., Acta Crystallogr D Biol Crystallogr, 52 pp. 758-765 (1996)).Dissociation of the TTR-tetramer at the T₄-binding interface, whichgenerates dimers that rapidly dissociate into amyloidogenic monomers, isthe rate-limiting step during TTR misfolding and amyloid formation.

There are more than 100 known amyloidogenic mutations in TTR, whichsegregate into ethnic and geographic groupings (Saraiva et al., HumMutat 5:191-196 (1995); Connors et al., Amyloid 10:160-184 (2003)).Point mutations in TTR promote TTR amyloidogenesis either by loweringthe thermodynamic stability of tetrameric TTR or through decreasing thekinetic barrier for tetramer dissociation, or both (Connelly et al.,Curr Opin Struct Biol 20:54-62 (2010)). These mutations lead tohereditary TTR amyloidoses such as familial amyloid polyneuropathy (FAP)and familial amyloid cardiomyopathy (FAC), which are both autosomaldominant conditions with varying ages of onset and penetrance dependingon ethnic background. One of the clinically most important FAP-causingTTR mutations is the thermodynamically destabilized Val30Met TTR (V30M)(Coelho Curr Opin Neurol 9:355-359 (1996)). However, the slow rate oftetramer dissociation (comparable to that of wild type TTR) limits thesteady-state concentration of the destabilized amyloidogenic monomer,which might explain the incomplete penetrance of the V30M mutation innon-Portuguese populations. The most common TTR variant with almostexclusive cardiac involvement, is the kinetically destabilizing V122Imutation. Although the V122I-TTR monomer has similar stability to thewild type (WT-TTR) monomer, the tetramer dissociates 3-fold faster underphysiological conditions and may explain the higher penetrance of theV122I mutation (Jiang et al., Proc Natl Acad Sci USA 98:14943-14948(2001)). This allele occurs in 3-4% of African-Americans (˜1.3 millionpeople) and is hypothesized to contribute to the increased prevalence ofheart failure among African Americans (Jacobson et al., N Engl J Med336:466-473 (1997); Connors et al. Am Heart J 158:607-614 (2009);Buxbaum et al. Am Heart J 159:864-870 (2010)). WT-TTR aggregationunderlies the development of senile systemic amyloidosis (SSA), acondition that affects up to 10-20% of the population over age 65.

Protein-protein interactions (PPIs) are a key regulatory mechanism for anumber of physiological and pathological cellular processes making themprime targets for therapeutic intervention (Arkin et al, Nat Rev DrugDiscov., 2004 Apr. 3(4):301-17). Although therapeutic antibodies thatblock PPIs are the fastest growing segment of the prescription-drugmarket, no small-molecule therapeutics have yet been approved for thisimportant target class. The majority of PPIs involve large interfaces,in many cases larger than 1500 Å² (up to 4500 Å²) in which the affinityis obtained by a multitude of often weak interactions. As a consequencethese widely spaced interactions are difficult to mimic with smallmolecules. Antagonizing PPIs with small, organic compounds is achallenging for a number of reasons: 1) a large, often flat, surfacearea is buried on each side of the interface, 2) the lack of deepcavities, 3) the nature of typical small-molecule libraries. Thus manyPPIs have come to be considered ‘undruggable’. Many extracellular PPItargets have been validated through the use of antibodies or otherprotein antagonists, some of which have become highly successful drugs.Examples are TNFa, IL2, IL4, IL13, VEGF, IFNa, SDF-1, CD4, MET, HER1&2.

The development of fluorescent and/or luminescent biosensors has emergedas a powerful tool for monitoring biomolecules in vitro and in vivo.Förster resonance energy transfer (FRET) is a method that measuresdistance-dependent energy transfer between two chromophores, one thedonor and the other the acceptor (Gell et al, Adv Exp Med Biol. 2012;747:19-41.; Miyawaki A., Annu Rev Biochem. 2011 Jun. 7; 80:357-73). Thedonor, after excitation by light, can transfer energy to the acceptorvia an induced dipole induced dipole interaction. The efficiency of theenergy transfer depends on the sixth power of the distance between thedyes. In general, the acceptor must be at 10-80 A distance to the donorfor efficient energy transfer. FRET is a useful technique becausemeasurements can be under physiological conditions and is one of themost widely used sensing mechanisms for ratiometric fluorescent probes.It permits the investigation of protein/protein interactions and hasproven to be a robust method for the investigation of the dynamics ofprotein complex composition and stoichiometry.

SUMMARY

Disclosed herein are compounds and compositions thereof, which find usein increasing the stability of proteins, particularly proteins that maymisfold and form aggregates. Also provided herein are methods for usingthese compounds and compositions for increasing stability of proteinsand thereby decreasing aggregate formation by these proteins. Alsodisclosed herein are heterobifunctional compounds that include a TTRbinding compound connected to a targeting moiety via a linker, for usein disrupting PPIs of a target protein. Also disclosed herein arelabeled compounds binding to the T₄ pocket of TTR, which are used todetermine the concentration of stabilized and/or tetrameric TTR, eitherby measurement of retained label and/or by measurement ofdistance-dependent energy transfer between the labeled compound bound tothe T₄ binding pocket and a labeled compound and/or peptide and/orprotein bound to an orthogonal binding surface on the TTR tetramer.

Provided herein are methods for using the disclosed compounds toincrease the stability of tetrameric TTR thereby preventing tetramerdissociation leading to TTR dimer and monomer misfolding, proteinaggregation and the formation and deposition of TTR amyloid.

The TTR stabilizers disclosed herein may be used to decrease TTR amyloidformation and/or to decrease cell dysfunction and/or death associatedwith TTR amyloid formation. The TTR stabilizers may be used to decreaseTTR amyloid formation in vitro in a cell-free system, in vitro- intra orextracellularily in cell culture, and in vivo, such as TTR found inbodily fluids, including but not restricted to blood, serum,cerebrospinal fluid, tissue and organs, including but not restricted to,the heart, the kidney, peripheral nerves, meninges, the central nervoussystem, the eye (including the retina and vitreous fluid) of a subject.As such, methods for using the disclosed compounds include administeringthe disclosed compounds in vitro, ex vivo or to a subject in vivo toincrease the stability of TTR found in bodily fluids, including but notrestricted to blood, serum, cerebrospinal fluid, tissues and organs,including but not restricted to, the heart, the kidney, peripheralnerves, meninges, the central nervous system, the eyes.

Also provided herein are methods for the treatment, prevention, delay orimprovement of one or more symptoms of TTR amyloidoses as well asmethods for the administration of a therapeutically effective amount ofa compound provided herein. In one embodiment, the compound preventsdissociation of a transthyretin tetramer by stabilization of the TTRtetramer. The TTR amyloid diseases include, but are not limited tofamilial amyloid polyneuropathy, familial amyloid cardiomyopathy,leptomeningeal amyloidis, oculoleptomengial amyloidosis, senile systemicamyloidosis, vitreous amyloidosis, CNS amyloidsis. Other amyloiddiseases include but are not restricted to ocular amyloidosis,gastrointestinal amyloidoses, neuropathic amyloidoses, non-neuropathicamyloidoses, nephropathy, non-hereditary amyloidoses, reactive/secondaryamyloidoses, cerebral amyloidoses, Alzheimer's disease, spongiformencephalopathy (i.e. Creutzfeldt Jakob disease, GSS, fatal familialinsomnia), frontotemporal dementia, Parkinson's disease, amyotrophiclateral sclerosis (ALS), Down Syndrome, multiple sclerosis,polyneuropathy, Guillain-Barre' syndrome, macular degeneration, vitreousopacities, glaucoma, type II diabetes and medullary carcinoma of thethyroid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the evaluation of ligands binding (Compound VIIc,Compound A, and tafamidis) to TTR in buffer by fluorescencepolarization.

FIG. 1 depicts TTR ligands (10 μM) competitively displace the FP-probe 5(0.2 μM) from TTR (0.4 μM) through binding to the T₄-binding sites (thelower the probe binding, the higher the binding affinity of TTR ligand).

FIG. 2 depicts the competition of FP-probe 5 from TTR by increasingconcentrations (0.003 to 100 μM) of Compound VIIc (K_(app)=193 nM,R²=0.994) and tafamidis (K_(app)=247 nM, R²=0.990). Each point shows themean+/−SD of three replicates.

FIG. 3 depicts the assessment of the binding affinity of Compound VIIc,Compound A, and tafamidis to TTR by isothermal titration calorimetry.(A) Calorimetric titration of Compound VIIc against TTR (K_(d1)=4.8 nMand K_(d2)=314 nM). (B) Calorimetric titration of Compound A against TTR(K_(d1)=58 nM and K_(d2)=500 nM). (C) Calorimetric titration oftafamidis against TTR (K_(d1)=4.4 nM and K_(d2)=280 nM). Raw data (top)and integrated heats (bottom) from the titration of TTR (2 μM) with testcompounds (25 μM).

FIG. 4 depicts fluorescence change due to modification of TTR in humanserum by covalent probe 6 (A) monitored for 6 h in the presence of probealone (black circles) or probe and TTR ligands (Compound VIIc, CompoundA, and tafamidis) (colors) (B) percentage of covalent probe 6 binding toTTR in the presence of ligands (Compound VIIc, Compound A, andtafamidis) measured after 6 hours of incubation, relative to probe alone(The lower the binding of the probe the higher the binding selectivityof the ligand to TTR). Each bar shows the mean+/−SD of three replicates.

FIG. 5 depicts the stabilization of WT-TTR in serum againstacid-mediated denaturation in (A) the presence of 10 microM CompoundVIIc, Compound A, and tafamidis, (B) the presence of concentration range(0.1 to 10 microM) of Compound VIIc and Tafamidis. Serum samples wereincubated in acetate buffer (pH 4.0), with DMSO or M of test compound,for the desired time period (0 and 72 hours) before cross-linking,SDS-PAGE, and immunoblotting. The density of all TTR bands (TTRtetramer; arrowhead and TTR bound to RBP; solid arrow) was quantifiedusing an Odyssey infrared imaging system (LI-COR Bioscience, Lincoln,NE) and reported as % TTR tetramer=100×[(tetramer & tetramer+RBPdensity, 72 hrs)/(tetramer & tetramer+RBP density of DMSO, 0 hrs)]. Eachbar shows the mean+/−SD of three replicates.

FIGS. 6A-6B illustrate the stabilization of V122I-TTR in serum from twoFAC patients against acid-mediated denaturation in the presence ofCompound VIIc and tafamidis. Serum samples were incubated in acetatebuffer (pH 4.0), with DMSO or 10 μM of tafamidis and Compound VIIc, forthe desired time period (0 and 72 hours) before cross-linking, SDS-PAGE,and immunoblotting. Each bar shows the mean+/−SD of three replicates.

FIG. 7 depicts the assessment of the cytotoxicity of Compound VIIc,Compound A, and tafamidis, at concentrations from 1 to 100 μM, on fourdifferent cell lines: Hep3B: human hepatoma cell line; Jurkat: Tlymphocyte cell line; MCF7: breast cancer cell line HeLa: cervicalcancer cell line; Cell viability was assessed using the MTT assay after24 h. Cell viability results are reported relative to cells treated withvehicle only (100% cell viability). Each bar shows the mean+/−SD ofthree replicates.

DEFINITIONS

“In combination with” as used herein refers to uses where, for example,the first compound is administered during the entire course ofadministration of the second compound; where the first compound isadministered for a period of time that is overlapping with theadministration of the second compound, e.g. where administration of thefirst compound begins before the administration of the second compoundand the administration of the first compound ends before theadministration of the second compound ends; where the administration ofthe second compound begins before the administration of the firstcompound and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe first compound begins before administration of the second compoundbegins and the administration of the second compound ends before theadministration of the first compound ends; where the administration ofthe second compound begins before administration of the first compoundbegins and the administration of the first compound ends before theadministration of the second compound ends. As such, “in combination”can also refer to regimen involving administration of two or morecompounds. “In combination with” as used herein also refers toadministration of two or more compounds which may be administered in thesame or different formulations, by the same of different routes, and inthe same or different dosage form type.

The term “isolated compound” means a compound which has beensubstantially separated from, or enriched relative to, other compoundswith which it occurs in nature or during chemical synthesis. Isolatedcompounds are usually at least about 80% pure, or at least about 90%pure, at least about 98% pure, or at least about 99% pure, by weight.The present invention is meant to encompass diastereomers as well astheir racemic and resolved, enantiomerically pure forms andpharmaceutically acceptable salts thereof.

“Treating” or “treatment” of a condition or disease includes: (1)preventing, ameliorating or altering at least one symptom of theconditions in a beneficial manner, i.e., causing a clinical symptom tonot significantly develop in a mammal that may be exposed to orpredisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, i.e., arresting orreducing the development of the disease and/or its symptoms, or (3)relieving the disease, i.e., causing regression or cure of the diseaseor its clinical symptoms. As used herein, amelioration of the symptomsof a particular disorder by administration of a particular compound orpharmaceutical composition refers to any lessening of disease symptomsand/or progression, whether permanent or temporary or lasting ortransient, that is or can be attributed to or associated with theadministration of the subject compound or composition.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound that, when administered to a mammal or othersubject for treating a disease, is sufficient to effect such treatmentfor the disease. The “therapeutically effective amount” will varydepending on the compound, the disease and its severity and the age,weight, etc., of the subject to be treated.

The terms “subject” and “patient” mean a mammal that may have a need forthe pharmaceutical methods, compositions and treatments describedherein. Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), and other subjects. Humans and non-human animalshaving commercial importance (e.g., livestock and domesticated animals)are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier or vehicle. The specifications for the novel unitdosage forms of the present invention depend on the particular compoundemployed and the effect to be achieved, and the pharmacodynamicsassociated with each compound in the host.

The term “physiological conditions” is meant to encompass thoseconditions compatible with living cells, e.g., predominantly aqueousconditions of a temperature, pH, salinity, etc. that are compatible withliving cells.

A “pharmaceutically acceptable excipient,” “pharmaceutically acceptablediluent,” “pharmaceutically acceptable carrier,” and “pharmaceuticallyacceptable adjuvant” means an excipient, diluent, carrier, and adjuvantthat are useful in preparing a pharmaceutical composition that aregenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and include an excipient, diluent, carrier, and adjuvantthat are acceptable for veterinary use as well as human pharmaceuticaluse. “A pharmaceutically acceptable excipient, diluent, carrier andadjuvant” as used in the specification and claims includes both one andmore than one such excipient, diluent, carrier, and adjuvant.

As used herein, a “pharmaceutical composition” is meant to encompass acomposition suitable for administration to a subject, such as a mammal,especially a human. In general a “pharmaceutical composition” ispreferably sterile, and free of contaminants that are capable ofeliciting an undesirable response within the subject (e.g., thecompound(s) in the pharmaceutical composition is pharmaceutical grade).Pharmaceutical compositions can be designed for administration tosubjects or patients in need thereof via a number of different routes ofadministration including oral, buccal, rectal, parenteral,intraperitoneal, intradermal, intracheal and the like.

As used herein, “pharmaceutically acceptable derivatives” of a compoundof the invention include salts, esters, enol ethers, enol esters,acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases,solvates, hydrates or prodrugs thereof. Such derivatives may be readilyprepared by those of skill in this art using known methods for suchderivatization. The compounds produced may be administered to animals orhumans without substantial toxic effects and either are pharmaceuticallyactive or are prodrugs.

As used herein, pharmaceutically acceptable derivatives of a compoundinclude salts, esters, enol ethers, enol esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, solvates, hydrates or prodrugsthereof. Such derivatives may be readily prepared by those of skill inthis art using known methods for such derivatization. The compoundsproduced may be administered to animals or humans without substantialtoxic effects and either are pharmaceutically active or are prodrugs.

A “pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. Such salts include: (1)acid addition salts, formed with inorganic acids such as hydrochloricacid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, andthe like; or formed with organic acids such as acetic acid, propionicacid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvicacid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,4,4′-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuricacid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylicacid, stearic acid, muconic acid, and the like; or (2) salts formed whenan acidic proton present in the parent compound either is replaced by ametal ion, e.g., an alkali metal ion, an alkaline earth ion, or analuminum ion; or coordinates with an organic base such as ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine, andthe like.

Pharmaceutically acceptable salts may also include, but are not limitedto, amine salts, such as but not limited toN,N′-dibenzylethylenediamine, chloroprocaine, choline, ammonia,diethanolamine and other hydroxyalkylamines, ethylenediamine,N-methylglucamine, procaine, N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Other pharmaceuticallyacceptable salts include acid salts such as acetate, adipate, alginate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate,camphorate, camphorsulfonate, cyclopentanepropionate, digluconate,dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate;base salts including ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth. Also, basic nitrogen-containing groupscan be quaternized with such agents as lower alkyl halides, such asmethyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides, such as benzyl and phenethylbromides and others. Water or oil-soluble or dispersible products arethereby obtained.

Pharmaceutically acceptable esters include, but are not limited to,alkyl, alkenyl, alkynyl, and cycloalkyl esters of acidic groups,including, but not limited to, carboxylic acids, phosphoric acids,phosphinic acids, sulfonic acids, sulfinic acids and boronic acids.

Pharmaceutically acceptable enol ethers include, but are not limited to,derivatives of formula C═C(OR) where R is hydrogen, alkyl, alkenyl,alkynyl, or cycloalkyl. Pharmaceutically acceptable enol esters include,but are not limited to, derivatives of formula C═C(OC(O)R) where R ishydrogen, alkyl, alkenyl, alkynyl, or cycloalkyl. Pharmaceuticallyacceptable solvates and hydrates are complexes of a compound with one ormore solvent or water molecules, or 1 to about 100, or 1 to about 10, orone to about 2, 3 or 4, solvent or water molecules.

A “pharmaceutically acceptable solvate or hydrate” of a compound of theinvention means a solvate or hydrate complex that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound, and includes, but is not limited to, complexes of acompound of the invention with one or more solvent or water molecules,or 1 to about 100, or 1 to about 10, or one to about 2, 3 or 4, solventor water molecules.

The term “organic group” and “organic radical” as used herein means anycarbon-containing group, including hydrocarbon groups that areclassified as an aliphatic group, cyclic group, aromatic group,functionalized derivatives thereof and/or various combination thereof.The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group and encompasses alkyl, alkenyl, and alkynylgroups, for example. The term “alkyl group” means a substituted orunsubstituted, saturated linear or branched hydrocarbon group or chain(e.g., C₁ to C₈) including, for example, methyl, ethyl, isopropyl,tert-butyl, heptyl, iso-propyl, n-octyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. Suitable substituents include carboxy,protected carboxy, amino, protected amino, halo, hydroxy, protectedhydroxy, nitro, cyano, monosubstituted amino, protected monosubstitutedamino, disubstituted amino, C₁ to C₇ alkoxy, C₁ to C₇ acyl, C₁ to C₇acyloxy, and the like. The term “substituted alkyl” means the abovedefined alkyl group substituted from one to three times by a hydroxy,protected hydroxy, amino, protected amino, cyano, halo, trifloromethyl,mono-substituted amino, di-substituted amino, lower alkoxy, loweralkylthio, carboxy, protected carboxy, or a carboxy, amino, and/orhydroxy salt. As used in conjunction with the substituents for theheteroaryl rings, the terms “substituted (cycloalkyl)alkyl” and“substituted cycloalkyl” are as defined below substituted with the samegroups as listed for a “substituted alkyl” group. The term “alkenylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon double bonds, such as a vinyl group. The term“alkynyl group” means an unsaturated, linear or branched hydrocarbongroup with one or more carbon-carbon triple bonds. The term “cyclicgroup” means a closed ring hydrocarbon group that is classified as analicyclic group, aromatic group, or heterocyclic group. The term“alicyclic group” means a cyclic hydrocarbon group having propertiesresembling those of aliphatic groups. The term “aromatic group” or “arylgroup” means a mono- or polycyclic aromatic hydrocarbon group, and mayinclude one or more heteroatoms, and which are further defined below.The term “heterocyclic group” means a closed ring hydrocarbon in whichone or more of the atoms in the ring are an element other than carbon(e.g., nitrogen, oxygen, sulfur, etc.), and are further defined below.

“Organic groups” may be functionalized or otherwise comprise additionalfunctionalities associated with the organic group, such as carboxyl,amino, hydroxyl, and the like, which may be protected or unprotected.For example, the phrase “alkyl group” is intended to include not onlypure open chain saturated hydrocarbon alkyl substituents, such asmethyl, ethyl, propyl, t-butyl, and the like, but also alkylsubstituents bearing further substituents known in the art, such ashydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino,carboxyl, etc. Thus, “alkyl group” includes ethers, esters, haloalkyls,nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, etc.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo groups. There can be one or more halogen, which are the same ordifferent. Halogens of particular interest include fluoro, chloro andbromo groups.

The term “haloalkyl” refers to an alkyl group as defined above that issubstituted by one or more halogen atoms. The halogen atoms may be thesame or different. The term “dihaloalkyl” refers to an alkyl group asdescribed above that is substituted by two halo groups, which may be thesame or different. The term “trihaloalkyl” refers to an alkyl group asdescribe above that is substituted by three halo groups, which may bethe same or different. The term “perhaloalkyl” refers to a haloalkylgroup as defined above wherein each hydrogen atom in the alkyl group hasbeen replaced by a halogen atom. The term “perfluoroalkyl” refers to ahaloalkyl group as defined above wherein each hydrogen atom in the alkylgroup has been replaced by a fluoro group.

The term “cycloalkyl” means a mono-, bi-, or tricyclic saturated ringthat is fully saturated or partially unsaturated. Examples of such agroup included cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin,bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl,1,4-cyclooctadienyl, and the like.

The term “(cycloalkyl)alkyl” means the above-defined alkyl groupsubstituted for one of the above cycloalkyl rings. Examples of such agroup include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl,5-(cyclopentyl)hexyl, 6-(adamantyl)hexyl, and the like.

The term “substituted phenyl” specifies a phenyl group substituted withone or more moieties, and in some instances one, two, or three moieties,chosen from the groups consisting of halogen, hydroxy, protectedhydroxy, cyano, nitro, trifluoromethyl, C₁ to C₇ alkyl, C₁ to C₇ alkoxy,C₁ to C₇ acyl, C₁ to C₇ acyloxy, carboxy, oxycarboxy, protected carboxy,carboxymethyl, protected carboxymethyl, hydroxymethyl, protectedhydroxymethyl, amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, carboxamide, protectedcarboxamide, N—(C₁ to C₆ alkyl)carboxamide, protected N—(C₁ to C₆alkyl)carboxamide, N,N-di(C₁ to C₆ alkyl)carboxamide, trifluoromethyl,N—((C₁ to C₆ alkyl)sulfonyl)amino, N-(phenylsulfonyl)amino or phenyl,substituted or unsubstituted, such that, for example, a biphenyl ornaphthyl group results.

Examples of the term “substituted phenyl” includes a mono- ordi(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl,2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl,3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl andthe like; a mono or di(hydroxy)phenyl group such as 2, 3, or4-hydroxyphenyl, 2,4-dihydroxyphenyl, the protected-hydroxy derivativesthereof and the like; a nitrophenyl group such as 2, 3, or4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4-cyanophenyl;a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl,2,4-dimethylphenyl, 2, 3 or 4-(iso-propyl)phenyl, 2, 3, or4-ethylphenyl, 2, 3 or 4-(n-propyl)phenyl and the like; a mono ordi(alkoxy)phenyl group, for example, 2,6-dimethoxyphenyl, 2, 3 or4-(isopropoxy)phenyl, 2, 3 or 4-(t-butoxy)phenyl,3-ethoxy-4-methoxyphenyl and the like; 2, 3 or 4-trifluoromethylphenyl;a mono- or dicarboxyphenyl or (protected carboxy)phenyl group such as 2,3 or 4-carboxyphenyl or 2,4-di(protected carboxy)phenyl; a mono- ordi(hydroxymethyl)phenyl or (protected hydroxymethyl)phenyl such as 2, 3or 4-(protected hydroxymethyl)phenyl or 3,4-di(hydroxymethyl)phenyl; amono- or di(aminomethyl)phenyl or (protected aminomethyl)phenyl such as2, 3 or 4-(aminomethyl)phenyl or 2,4-(protected aminomethyl)phenyl; or amono- or di(N-(methylsulfonylamino))phenyl such as 2, 3 or4-(N-(methylsulfonylamino))phenyl. Also, the term “substituted phenyl”represents disubstituted phenyl groups wherein the substituents aredifferent, for example, 3-methyl-4-hydroxyphenyl,3-chloro-4-hydroxyphenyl, 2-methoxy-4-bromophenyl,4-ethyl-2-hydroxyphenyl, 3-hydroxy-4-nitrophenyl,2-hydroxy-4-chlorophenyl and the like.

The term “(substituted phenyl)alkyl” means one of the above substitutedphenyl groups attached to one of the above-described alkyl groups.Examples of include such groups as 2-phenyl-1-chloroethyl,2-(4′-methoxyphenyl)ethyl, 4-(2′,6′-dihydroxy phenyl)n-hexyl,2-(5′-cyano-3′-methoxyphenyl)n-pentyl, 3-(2′,6′-dimethylphenyl)n-propyl,4-chloro-3-aminobenzyl, 6-(4′-methoxyphenyl)-3-carboxy(n-hexyl),5-(4′-aminomethylphenyl)-3-(aminomethyl)n-pentyl,5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxynapth-2-yl)methyl and the like.

As noted above, the term “aromatic” or “aryl” refers to six memberedcarbocyclic rings. Also as noted above, the term “heteroaryl” denotesoptionally substituted five-membered or six-membered rings that have 1to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, inparticular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms.

Furthermore, the above optionally substituted five-membered orsix-membered rings can optionally be fused to an aromatic 5-membered or6-membered ring system. For example, the rings can be optionally fusedto an aromatic 5-membered or 6-membered ring system such as a pyridineor a triazole system, and preferably to a benzene ring.

The following ring systems are examples of the heterocyclic (whethersubstituted or unsubstituted) radicals denoted by the term “heteroaryl”:thienyl, furyl, pyrrolyl, pyrrolidinyl, imidazolyl, isoxazolyl,triazolyl, thiadiazolyl, oxadiazolyl, tetrazolyl, thiatriazolyl,oxatriazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl,triazinyl, thiadiazinyl tetrazolo, 1,5-[b]pyridazinyl and purinyl, aswell as benzo-fused derivatives, for example, benzoxazolyl,benzthiazolyl, benzimidazolyl and indolyl.

Substituents for the above optionally substituted heteroaryl rings arefrom one to three halo, trihalomethyl, amino, protected amino, aminosalts, mono-substituted amino, di-substituted amino, carboxy, protectedcarboxy, carboxylate salts, hydroxy, protected hydroxy, salts of ahydroxy group, lower alkoxy, lower alkylthio, alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, (cycloalkyl)alkyl, substituted(cycloalkyl)alkyl, phenyl, substituted phenyl, phenylalkyl, and(substituted phenyl)alkyl. Substituents for the heteroaryl group are asheretofore defined, or in the case of trihalomethyl, can betrifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl. Asused in conjunction with the above substituents for heteroaryl rings,“lower alkoxy” means a C₁ to C⁴ alkoxy group, similarly, “loweralkylthio” means a C₁ to C₄ alkylthio group.

The term “(monosubstituted)amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, alkyl, substituted alkyl, C₁ to C₄ acyl, C₂ to C₇ alkenyl, C₂ toC₇ substituted alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇ to C₁₆substituted alkylaryl and heteroaryl group. The (monosubstituted) aminocan additionally have an amino-protecting group as encompassed by theterm “protected (monosubstituted)amino.” The term “(disubstituted)amino”refers to amino groups with two substituents chosen from the groupconsisting of phenyl, substituted phenyl, alkyl, substituted alkyl, C₁to C₇ acyl, C₂ to C₇ alkenyl, C₂ to C₇ alkynyl, C₇ to C₁₆ alkylaryl, C₇to C₁₆ substituted alkylaryl and heteroaryl. The two substituents can bethe same or different.

The term “heteroaryl(alkyl)” denotes an alkyl group as defined above,substituted at any position by a heteroaryl group, as above defined.

“Optional” or “optionally” means that the subsequently described event,circumstance, feature or element may, but need not, occur, and that thedescription includes instances where the event or circumstance occursand instances in which it does not. For example, “heterocyclo groupoptionally mono- or di-substituted with an alkyl group” means that thealkyl may, but need not, be present, and the description includessituations where the heterocyclo group is mono- or disubstituted with analkyl group and situations where the heterocyclo group is notsubstituted with the alkyl group.

Compounds that have the same molecular formula but differ in the natureor sequence of bonding of their atoms or the arrangement of their atomsin space are termed “isomers.” Isomers that differ in the arrangement oftheir atoms in space are termed “stereoisomers.” Stereoisomers that arenot mirror images of one another are termed “diastereomers” and thosethat are non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, itis bonded to four different groups, a pair of enantiomers is possible.An enantiomer can be characterized by the absolute configuration of itsasymmetric center and is described by the R- and S-sequencing rules ofCahn and Prelog, or by the manner in which the molecule rotates theplane of polarized light and designated as dextrorotatory orlevorotatory (i.e., as (+) or (−)-isomers respectively). A chiralcompound can exist as either individual enantiomer or as a mixturethereof. A mixture containing equal proportions of the enantiomers iscalled a “racemic mixture.”

The compounds of this invention may possess one or more asymmetriccenters; such compounds can therefore be produced as individual (R)- or(S)-stereoisomers or as mixtures thereof. Unless indicated otherwise,the description or naming of a particular compound in the specificationand claims is intended to include both individual enantiomers andmixtures, racemic or otherwise, thereof. The methods for thedetermination of stereochemistry and the separation of stereoisomers arewell-known in the art (see, e.g., the discussion in Chapter 4 of“Advanced Organic Chemistry”, 4th edition J. March, John Wiley and Sons,New York, 1992).

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001.

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as high performanceliquid chromatography (HPLC), preparative thin layer chromatography,flash column chromatography and ion exchange chromatography. Anysuitable stationary phase can be used, including normal and reversedphases as well as ionic resins. See, e.g., Introduction to Modern LiquidChromatography, 2nd Edition, ed. L. R. Snyder and J. J. Kirkland, JohnWiley and Sons, 1979; and Thin Layer Chromatography, ed E. Stahl,Springer-Verlag, New York, 1969.

During any of the processes for preparation of the compounds of thepresent disclosure, it may be necessary and/or desirable to protectsensitive or reactive groups on any of the molecules concerned. This canbe achieved by means of conventional protecting groups as described instandard works, such as T. W. Greene and P. G. M. Wuts, “ProtectiveGroups in Organic Synthesis”, Fourth edition, Wiley, New York 2006. Theprotecting groups can be removed at a convenient subsequent stage usingmethods known from the art.

The compounds described herein can contain one or more chiral centersand/or double bonds and therefore, can exist as stereoisomers, such asdouble-bond isomers (i.e., geometric isomers), enantiomers ordiastereomers. Accordingly, all possible enantiomers and stereoisomersof the compounds including the stereoisomerically pure form (e.g.,geometrically pure, enantiomerically pure or diastereomerically pure)and enantiomeric and stereoisomeric mixtures are included in thedescription of the compounds herein. Enantiomeric and stereoisomericmixtures can be resolved into their component enantiomers orstereoisomers using separation techniques or chiral synthesis techniqueswell known to the skilled artisan. The compounds can also exist inseveral tautomeric forms including the enol form, the keto form andmixtures thereof. Accordingly, the chemical structures depicted hereinencompass all possible tautomeric forms of the illustrated compounds.The compounds described also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that can beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Compounds canexist in unsolvated forms as well as solvated forms, including hydratedforms. In general, compounds can be hydrated or solvated. Certaincompounds can exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplatedherein and are intended to be within the scope of the presentdisclosure.

DETAILED DESCRIPTION

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a method” includesa plurality of such methods and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the chemical groups represented by the variables (e.g.,-J, ═W—, —X═, ═Y—, —Z═, -Q, —R^(V1), —R^(V2), —R^(V3), —R^(V4), —R^(T),—R^(TT), -Q^(CA), -Q^(HA), —R^(PP), —R^(R), —R^(RA), -L^(R)-, -M^(R),—R^(K), —R^(RR), —R^(J), -M^(J), —R^(N), —R^(J1), —R^(J2), —R^(J3),—R^(J4), —R^(J5), —R^(J6), -L^(J)-, —R^(J2X), —R^(J2XX), —R^(JJ),—R^(JJJ), -L^(JJJ)-, —R^(P2), —R^(P3), —R^(P4), —R^(P5), —R^(P6),—R^(P2R), —R^(P2RA), —R^(P3R), —R^(P3RA), —R^(P4R), —R^(P4RA), —R^(P5R),—R^(P5RA), —R^(P6R), —R^(P6RA), —R^(AK), etc.) are specifically embracedby the present invention and are disclosed herein just as if each andevery combination was individually and explicitly disclosed, to theextent that such combinations embrace compounds that are stablecompounds (i.e., compounds that can be isolated, characterized, andtested for biological activity). In addition, all sub-combinations ofthe chemical groups listed in the embodiments describing such variablesare also specifically embraced by the present invention and aredisclosed herein just as if each and every such sub-combination ofchemical groups was individually and explicitly disclosed herein

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Overview

The present disclosure is based on the identification of compounds thatbind to a TTR tetramer in the presence of a TTR ligand known to bind andstabilize TTR tetramer. These compounds stabilize TTR tetramers andthereby reduce the formation of TTR amyloid. These compounds also finduse to determine the level of stabilized and/or tetrameric TTR. Inaddition, these compounds find use in the preparation ofheterobifunctional compounds that recruit TTR for use in disruptingPPIs.

Compositions

Provided herein are compounds that may be used to stabilize TTRtetramers reducing TTR amyloid formation. These compounds can beincorporated into a variety of formulations for therapeuticadministration by a variety of routes, including but not limited tooral, parenteral, transdermal, intrathecal, ophthalmic, topical,pulmonary, nasal, rectal or depot administration.

More particularly, the compounds disclosed herein can be formulated intopharmaceutical compositions by combination with appropriate,pharmaceutically acceptable carriers, diluents, excipients and/oradjuvants.

Compounds

In some embodiments, a compound of the invention is of the structure ofCompound Ia:

-   -   where X_(a), X_(b), and X_(c) are independently selected from        C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are independently        selected from hydrogen, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, alkoxy, aryloxy, hydroxyl, a heterocyclic        group, halogen, nitro; acyl, substituted acyl, carboxyl,        alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   the A ring is a 4 to 12-membered ring, in certain embodiments        the 4 to 12-membered ring is an aromatic or heteroaromatic ring;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano;    -   c is a number from zero to 5; and    -   the B ring is a heterocyclic ring selected from the following        (h1-h30):

-   -   where R¹¹-R¹⁶ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino, and cyano;        and R¹⁷ is selected from a hydroxyl, alkyl, amino, and alkyl        amino;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound Ib:

-   -   where R¹ and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, aryl,        substituted aryl, alkoxy, aryloxy, hydroxyl, a heterocyclic        group, halogen, nitro; acyl, substituted acyl, carboxyl,        alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   R² is selected from hydrogen, alkyl, substituted alkyl, aryl,        substituted aryl, alkoxy, aryloxy, hydroxyl, a heterocyclic        group, acyl, substituted acyl, alkoxycarbonyl, substituted        alkoxycarbonyl, acylamino, substituted acylamino, thioalkyl and        cyano;    -   X_(a), X_(b) and X_(c) are independently selected from        C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are independently        selected from hydrogen, alkyl, substituted alkyl, alkenyl,        substituted alkenyl, alkynyl, substituted alkynyl, aryl,        substituted aryl, alkoxy, aryloxy, hydroxyl, a heterocyclic        group, halogen, nitro; acyl, substituted acyl, carboxyl,        alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   the A ring is a 4 to 12-membered ring, in certain embodiments        the 4 to 12-membered ring is an aromatic or heteroaromatic ring;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano; and    -   c is a number from zero to 5;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound IIa:

-   -   where n is 1 to 8;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   the A ring is a 5 to 12-membered ring, in certain embodiments        the 5 to 12-membered ring is an aromatic or heteroaromatic ring;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano; and    -   c is a number from zero to 5;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound IIb:

-   -   where n is zero to 7;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   the A ring is a 5 to 12-membered ring, in certain embodiments        the 5 to 12-membered ring is an aromatic ring;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano; and    -   c is a number from zero to 5;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound is of the structure of Compound IIIa:

-   -   where n is zero to 7;    -   Z is carbon or up to three of the five Z may be nitrogen with        the remaining being carbon;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano; and    -   c is a number from zero to 5;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound is of the structure of Compound IIIb:

-   -   where n is 1 to 8;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   each Y is independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halogen, nitro; acyl,        substituted acyl, carboxyl, alkoxycarbonyl, substituted        alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,        substituted amino, acylamino, substituted acylamino,        sulfonamide, sulfonyl fluoride, thioester and cyano; and    -   c is a number from zero to 5;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound is of the structure of Compound IIIb:

-   -   where n is 1 to 4,    -   R¹ is a is a short chain alkyl having 1 to 4 carbon atoms;    -   R² is hydrogen;    -   R³ is a is a short chain alkyl having 1 to 4 carbon atoms;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   each Y is independently selected from a halogen, acyl,        substituted acyl, carboxyl, heterocyclic group, alkoxycarbonyl        and substituted alkoxycarbonyl; and    -   c is 2;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, acetal, amide, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound IVa:

-   -   where n is 1 to 8;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halo, nitro; acyl, substituted        acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl,        aminoacyl, substituted aminoacyl, amino, substituted amino,        acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   R^(a) is CHO, COOH, COOR⁶, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂,        CONHSO₂Ar, CONHCH(R⁹)COOH, hydrogen, halogen, alkyl, substituted        alkyl, acyl, substituted acyl, carboxyl, heterocyclic group,        sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or        substituted alkoxycarbonyl;    -   R^(b) is CHO, COOH, COOR⁶, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂,        CONHSO₂Ar, CONHCH(R⁹)COOH, hydrogen, halogen, alkyl, substituted        alkyl, acyl, substituted acyl, carboxyl, heterocyclic group,        sulfonamide, sulfonyl fluoride, thioester, alkoxycarbonyl or        substituted alkoxycarbonyl;    -   R⁶ is alkyl, haloalkyl, cycloalkyl, or heterocyclyl;    -   R⁷ and R⁸ are each independently hydrogen, alkyl, alkenyl,        alkynyl, cycloalkyl, heterocyclyl, or heteroaryl; and    -   R⁹ is the side chain of a naturally occurring α-amino carboxylic        acid;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound V:

-   -   where n is zero to 7;    -   R¹, R² and R³ are independently selected from hydrogen, alkyl,        substituted alkyl, alkenyl, substituted alkenyl, alkynyl,        substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,        hydroxyl, a heterocyclic group, halo, nitro; acyl, substituted        acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl,        aminoacyl, substituted aminoacyl, amino, substituted amino,        acylamino, substituted acylamino, and cyano;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   R^(a) and R^(b) are independently selected from CHO, COOH,        COOR⁶, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂, CONHSO₂Ar,        CONHCH(R⁹)COOH, CONHSO₂R¹⁰, hydrogen, halogen, alkyl,        substituted alkyl, acyl, substituted acyl, carboxyl,        heterocyclic group, sulfonamide, sulfonyl fluoride, thioester,        alkoxycarbonyl or substituted alkoxycarbonyl;    -   R^(c) and R^(d) are independently selected from hydrogen,        halogen, alkyl, substituted alkyl, acyl, substituted acyl,        carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride,        thioester, trifluoromethyl, amino, substituted amino, sulfonyl,        substituted sulfonyl, hydroxyl, alkoxycarbonyl or substituted        alkoxycarbonyl;    -   R⁶ is alkyl, haloalkyl, cycloalkyl, or heterocyclyl;    -   R⁷, R⁸ and R¹⁰ are each independently hydrogen, alkyl, alkenyl,        alkynyl, cycloalkyl, heterocyclyl, or heteroaryl; and    -   R⁹ is the side chain of a naturally occurring α-amino carboxylic        acid;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound VI:

-   -   where n is 2;    -   R¹ is a is a short chain alkyl having 1 to 4 carbon atoms;    -   R² is hydrogen;    -   R³ is a is a short chain alkyl having 1 to 4 carbon atoms;    -   X_(a) is C(R⁴)(R⁵), O, N—R⁵ or S; where R⁴ and R⁵ are        independently selected from hydrogen, alkyl, substituted alkyl,        alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,        aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, a        heterocyclic group, halogen, nitro; acyl, substituted acyl,        carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,        substituted aminoacyl, amino, substituted amino, acylamino,        substituted acylamino, and cyano;    -   R^(a) is CHO, COOH, COOR⁶, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂,        CONHSO₂Ar, CONHCH(R⁹)COOH, hydrogen, an acyl, substituted acyl,        carboxyl, heterocyclic group, sulfonamide, sulfonyl fluoride,        thioester, alkoxycarbonyl and substituted alkoxycarbonyl;    -   R^(b) is CHO, COOH, COOR⁶, CONR⁷R⁸, tetrazolyl, CONHOH, B(OH)₂,        CONHSO₂Ar, CONHCH(R⁹)COOH, a halogen, heterocyclic group, or        hydrogen;    -   R⁶ is alkyl, haloalkyl, cycloalkyl, or heterocyclyl;    -   R⁷ and R⁸ are each independently hydrogen, alkyl, alkenyl,        alkynyl, cycloalkyl, heterocyclyl, or heteroaryl; and    -   R⁹ is the side chain of a naturally occurring α-amino carboxylic        acid;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound VIIa:

-   -   where R^(a) is OH, CHO, COOH, CONH₂, CONH(OH), COOR⁶, CONHR⁶;        and    -   R⁶ is straight of branched alkyl of 1-3 carbon atoms;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound VIIb:

-   -   where R^(a) is COOH, CONH₂, CONH(OH), COOR⁶, CONHR⁶; and    -   R⁶ is straight of branched alkyl of 1-3 carbon atoms;    -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

In some embodiments, a compound of the invention is of the structure ofCompound VIIc:

-   -   or a pharmaceutically acceptable salt, ester, enol ether, enol        ester, amide, acetal, ketal, orthoester, hemiacetal, hemiketal,        hydrate, solvate or prodrug thereof.

Bifunctional Compounds for Disrupting PPIs

Also provided are heterobifunctional compounds that include arecruitment moiety connected to a targeting moiety via a linker. Therecruitment moiety is a ligand of an abundant serum protein (e.g., a TTRbinding compound of the disclosure, as described above). The targetingmoiety is a ligand for a protein target of interest. In someembodiments, the protein target is involved in a protein proteininteraction (PPI) where disruption of the PPI is desirable. For example,the PPI may be important in the regulation of a biological process thatleads to a particular disease condition, where disrupting the PPI ofinterest may provide a method of inhibiting or treating the diseasecondition.

In some embodiments, the heterobifunctional compound is of the formulaR-L-T, where the recruitment moiety R is a TTR-binding compounddescribed by a structure of Compounds Ia-VIIc, above; L is a linker, andT is a targeting moiety. In some cases, the subject heterobifunctionalcompound includes one or more, such as two or more, recruitmentmoieties.

The recruitment moiety is connected to the targeting moiety via alinker, at any convenient point of attachment, which may be readilyselected by one of ordinary skill in the art such that the bindingproperty of the ligand to its cognate protein is not significantlyreduced. In the TTR binding compounds described above, the position atwhich a linker may be connected using any convenient chemicalmodification chemistries is determined using any convenient selectionmethod, such as but not limited to, modeling a X-ray crystal structureof TTR (e.g., a co-crystal structure of TTR with a ligand) to determinethe mode of binding of the recruitment moiety to TTR and to select oneor more appropriate positions which are not involved in contacts withthe protein (e.g., solvent exposed positions), and which may be readilychemically modified. Further methods include determining whether amodification of interest has an adverse effect of the binding of therecruitment moiety to TTR using an in vitro binding assay.

Any convenient targeting moiety may be used. The targeting moiety may bea small molecule that targets a therapeutic protein target of interest.For example, the targeting moiety may be any convenient binder to aprotein of a target ligand/receptor pair, such as but not limited to,IL2/IL2Rα, TNFα/TNFR1, VEGF-VEGFR, CCL12-CXCR4, CD4-gp120, c-Met-HGF,and LFA-1-CD54. Suitable positions of the targeting moieties describedabove, to which a linker may be attached are selected using anyconvenient method, such as but not limited to, modeling methods using aX-ray crystal structure of the target protein (e.g., a co-crystalstructure of target protein bound with the ligand) to model the mode ofbinding of the targeting moiety and to select appropriate positions thatare not involved in contacts with the target protein (e.g., solventexposed positions), and which may be readily chemically modified. Forexample, co-crystal structures of the IL-2 and TNF-alpha ligands areavailable for use in selecting convenient sites in these targetingmoieties for chemical modification and attachment of linkers inpreparation of subject heterobifunctional compounds. Further methodsinclude determining whether a modification of interest has an adverseeffect of the binding of the targeting moiety to the target proteinusing an in vitro binding assay.

Exemplary modifications of IL-2 and TNF-α targeting moieties of interestthat may be used to attach targeting moieties to linkers inheterobifunctional compounds are shown below:

As used herein, the term “linker”, “linkage” and “linking group” refersto a linking moiety that connects two groups and has a backbone of 20atoms or less in length. A linker or linkage may be a covalent bond thatconnects two groups or a chain of between 1 and 20 atoms in length, forexample of about 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 or 20 carbonatoms in length, where the linker may be linear, branched, cyclic or asingle atom. In certain cases, one, two, three, four or five or morecarbon atoms of a linker backbone may be optionally substituted with asulfur, nitrogen or oxygen heteroatom. The bonds between backbone atomsmay be saturated or unsaturated, usually not more than one, two, orthree unsaturated bonds will be present in a linker backbone. The linkermay include one or more substituent groups, for example with an alkyl,aryl or alkenyl group. A linker may include, without limitations,oligo(ethylene glycol); ethers, thioethers, tertiary amines, alkyls,which may be straight or branched, e.g., methyl, ethyl, n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), and the like. The linker backbone may include a cyclic group,for example, an aryl, a heterocycle or a cycloalkyl group, where 2 ormore atoms, e.g., 2, 3 or 4 atoms, of the cyclic group are included inthe backbone. A linker may be cleavable or non-cleavable.

The linking moiety may be conjugated to the recruitment and targetingmoieties using any convenient functional groups (carboxylic acids,amines, alcohols, carbamates, esters, ethers, thioethers, maleimides,and the like), and linking chemistries. For example, any convenientconjugation chemistry described by G. T. Hermanson (“BioconjugateTechniques”, Academic Press, Second Edition, 2008) may be readilyadapted for use in preparing the subject heterobifunctional compounds.

Exemplary linkers that may be used in connecting the recruitment moietyto the targeting moiety using any convenient chemical modificationmethods are shown below:

Formulation of Pharmaceutical Compositions

The pharmaceutical compositions provided herein contain therapeuticallyeffective amounts of one or more of the compounds provided herein thatare useful in the prevention, treatment, or amelioration of one or moreof the symptoms of diseases or disorders associated with transthyretin(TTR) misfolding, or in which TTR misfolding is implicated, in apharmaceutically acceptable carrier. Diseases or disorders associatedwith TTR misfolding include, but are not limited to, familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, senile systemicamyloidosis, Alzheimer's disease, spongiform encephalopathy (i.e.,Creutzfeldt Jakob disease GSS, fatal familial insomnia), frontotemporaldementia, Parkinson's disease amyotrophic lateral sclerosis (ALS), DownSyndrome, multiple sclerosis, polyneuropathy, Guillain-Barre' syndrome,macular degeneration, vitreous opacities, glaucoma, type II diabetes ormedullary carcinoma of the thyroid. Pharmaceutical carriers suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

The compositions contain one or more compounds provided herein. Thecompounds are, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, thecompounds described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel Introduction to Pharmaceutical Dosage Forms, Fourth Edition1985, 126). In certain preferable embodiments, the compounds areformulated into suitable pharmaceutical preparations for oraladministration to a subject.

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof are mixed with asuitable pharmaceutical carrier. The compounds may be derivatized as thecorresponding salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs prior to formulation, as described above. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates oneor more of the symptoms of diseases or disorders associated with TTRmisfolding or in which TTR misfolding is implicated.

In certain embodiments, the compositions are formulated for singledosage administration. To formulate a composition, the weight fractionof compound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described and thenextrapolated therefrom for dosages for humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to ameliorate one or more of the symptoms ofdiseases or disorders associated with TTR misfolding or in which TTRmisfolding is implicated, as described herein.

In one embodiment, a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.1 ng/ml toabout 50-100 μg/ml. The pharmaceutical compositions, in anotherembodiment, should provide a dosage of from about 0.001 mg to about 2000mg of compound per kilogram of body weight per day. Pharmaceuticaldosage unit forms are prepared to provide from about 0.01 mg, 0.1 mg or1 mg to about 500 mg, 1000 mg or 2000 mg, and in one embodiment fromabout 10 mg to about 500 mg of the active ingredient or a combination ofessential ingredients per dosage unit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using surfactants, such as TWEEN™, or dissolution in aqueous sodiumbicarbonate. Derivatives of the compounds, such as prodrugs of thecompounds may also be used in formulating effective pharmaceuticalcompositions.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. For example, subject compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

Compositions for Oral Administration

Oral pharmaceutical dosage forms may be solid, gel or liquid. In certainembodiments, the solid dosage forms are tablets, capsules, granules, andbulk powders. Types of oral tablets include compressed, chewablelozenges and tablets which may be enteric-coated, sugar-coated orfilm-coated. Capsules may be hard or soft gelatin capsules, whilegranules and powders may be provided in non-effervescent or effervescentform with the combination of other ingredients known to those skilled inthe art.

Solid Compositions for Oral Administration

In certain embodiments, the formulations are solid dosage forms, in oneembodiment, capsules or tablets. The tablets, pills, capsules, trochesand the like can contain one or more of the following ingredients, orcompounds of a similar nature: a binder, a lubricant; a diluent; aglidant; a disintegrating agent; a coloring agent; a sweetening agent; aflavoring agent; a wetting agent; an emetic coating; and a film coating.Examples of binders include microcrystalline cellulose, gum tragacanth,glucose solution, acacia mucilage, gelatin solution, molasses,polyinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

The compound, or pharmaceutically acceptable derivative thereof, couldbe provided in a composition that protects it from the acidicenvironment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. Thecomposition may also be formulated in combination with an antacid orother such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The compounds can also be administeredas a component of an elixir, suspension, syrup, wafer, sprinkle, chewinggum or the like. A syrup may contain, in addition to the activecompounds, sucrose as a sweetening agent and certain preservatives, dyesand colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Liquid Compositions for Oral Administration

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examplesof preservatives include glycerin, methyl and propylparaben, benzoicacid, sodium benzoate and alcohol. Examples of non-aqueous liquidsutilized in emulsions include mineral oil and cottonseed oil. Examplesof emulsifying agents include gelatin, acacia, tragacanth, bentonite,and surfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include sodium carboxymethylcellulose, pectin, tragacanth, Veegumand acacia. Sweetening agents include sucrose, syrups, glycerin andartificial sweetening agents such as saccharin. Wetting agents includepropylene glycol monostearate, sorbitan monooleate, diethylene glycolmonolaurate and polyoxyethylene lauryl ether. Organic acids includecitric and tartaric acid. Sources of carbon dioxide include sodiumbicarbonate and sodium carbonate. Coloring agents include any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include natural flavors extracted from plants suchfruits, and synthetic blends of compounds which produce a pleasant tastesensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is in oneembodiment encapsulated in a gelatin capsule. Such solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545, the disclosures of which are hereinincorporated by reference. For a liquid dosage form, the solution, e.g.,for example, in a polyethylene glycol, may be diluted with a sufficientquantity of a pharmaceutically acceptable liquid carrier, e.g., water,to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603, the disclosures of which are herein incorporated byreference. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl)acetals of lower alkyl aldehydes such as acetaldehydediethyl acetal.

Injectables, Solutions and Emulsions

Parenteral administration, in one embodiment characterized by injection,either subcutaneously, intramuscularly or intravenously is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained is also contemplated herein.Briefly, a compound provided herein is dispersed in a solid innermatrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticizedor unplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include sodium chloride injection, Ringersinjection, isotonic dextrose injection, sterile water injection,dextrose and lactated Ringers injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcellulose,hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifyingagents include Polysorbate 80 (TWEENa 80). Sequestering or chelatingagents of metal ions include EDTA. Pharmaceutical carriers also includeethyl alcohol, polyethylene glycol and propylene glycol for watermiscible vehicles; and sodium hydroxide, hydrochloric acid, citric acidor lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active compound to thetreated tissue(s).

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Lyophilized Powders

Of interest herein are also lyophilized powders, which can bereconstituted for administration as solutions, emulsions and othermixtures. They may also be reconstituted and formulated as solids orgels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4.degree. C. to roomtemperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

Topical Administration

Topical mixtures are prepared as described for the local and systemicadministration. The resulting mixture may be a solution, suspension,emulsions or the like and are formulated as creams, gels, ointments,emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes,foams, aerosols, irrigations, sprays, suppositories, bandages, dermalpatches or any other formulations suitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation.These formulations for administration to the respiratory tract can be inthe form of an aerosol or solution for a nebulizer, or as a microfinepowder for insufflation, alone or in combination with an inert carriersuch as lactose. Where compositions are formulated as aerosols forinhalation or administration to the respiratory tract, the particles ofthe formulation may have diameters of 50 microns or less, such as 40microns or less, such as 30 microns or less, such as 25 microns or less,such as 15 microns or less, such as 10 microns or less, such as 5microns or less and including having diameters of 1 micron or less.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered.

These solutions, such as for example for ophthalmic use, may beformulated as 0.01%-10% isotonic solutions, pH about 5-7, withappropriate salts.

Compositions for Other Routes of Administration

Other routes of administration, such as transdermal patches, includingiontophoretic and electrophoretic devices, and buccal and rectaladministration, are also contemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devicesof interest may include, but are not limited to those disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010,715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, thedisclosures of which are herein incorporated by reference.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

Targeted Formulations

The compounds provided herein, or pharmaceutically acceptablederivatives thereof, may also be formulated to be targeted to aparticular tissue, receptor, or other area of the body of the subject tobe treated. Many such targeting methods are well known to those of skillin the art. All such targeting methods are contemplated herein for usein the instant compositions. Examples of targeting methods of interestmay include but are not limited to those described in U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874, thedisclosures of which are herein incorporated by reference.

In one embodiment, liposomal suspensions, including tissue-targetedliposomes, such as tumor-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art. For example, liposomeformulations may be prepared as described in U.S. Pat. No. 4,522,811,the disclosure of which is herein incorporated by reference. Briefly,liposomes such as multilamellar vesicles (MLV's) may be formed by dryingdown egg phosphatidyl choline and brain phosphatidyl serine (7:3 molarratio) on the inside of a flask. A solution of a compound providedherein in phosphate buffered saline lacking divalent cations (PBS) isadded and the flask shaken until the lipid film is dispersed. Theresulting vesicles are washed to remove unencapsulated compound,pelleted by centrifugation, and then resuspended in PBS.

Dosages of the Compounds of the Present Disclosure

Depending on the subject and condition being treated and on theadministration route, the subject compounds may be administered indosages of, for example, 0.1 μg to 10 mg/kg body weight per day. Therange is broad, since in general the efficacy of a therapeutic effectfor different mammals varies widely with doses typically being 20, 30 oreven 40 times smaller (per unit body weight) in man than in the rat.Similarly the mode of administration can have a large effect on dosage.Thus, for example, oral dosages may be about ten times the injectiondose. Higher doses may be used for localized routes of delivery.

A typical dosage may be a solution suitable for intravenousadministration; a tablet taken from one to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active ingredient, etc. Thetime-release effect may be obtained by capsule materials that dissolveat different pH values, by capsules that release slowly by osmoticpressure, or by any other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is one which provides up to about 1 μgto about 1,000 μg or about 10,000 μg of subject composition to reduce asymptom in a subject animal.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more compoundsof the invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the compound (s) in acomposition as a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

As discussed below, the present disclosure includes compounds andpharmaceutical compositions for stabilizing transthyretin and preventsdissociation of the transthyretin tetramer by kinetic stabilization ofthe native state of the transthyretin tetramer of TTR such as thosefound in the blood, serum, cerebrospinal fluid, fluids of the centralnervous system (CNS), retina and the eyes. As such, compounds andpharmaceutical compositions of interest include those, which arecompatible with the biological fluids of blood, serum, cerebrospinalfluid, fluids of the central nervous system, retina and the eyes.

Combination Therapy

For use in the subject methods, the subject compounds may be formulatedwith or otherwise administered in combination with otherpharmaceutically active agents, including other agents, which maymodulate intra or extracellular protein homeostasis and/or proteinstability and or protein aggregation and/or protein folding, such asresveratrol, heat shock proteins, protein chaperones, and mimicsthereof.

The compounds described above may also be administered in combinationwith other therapies for diseases caused by TTR amyloid. Therapies fordiseases caused by TTR amyloid include heart transplant for TTRcardiomyopathy, liver transplant, other kinetic stabilizers of TTR, RNAknock-down and/or RNA interference methods and the like. The compounddescribed above may be administered before, after, or during anothertherapy for diseases caused by TTR amyloid.

The compounds described herein for use in combination therapy with thecompounds of the present invention may be administered by the same routeof administration (e.g. intrapulmonary, oral, enteral, etc.) that thecompounds are administered. In the alternative, the compounds for use incombination therapy with the compounds of the present invention may beadministered by a different route of administration that the compoundsare administered.

The compounds and compositions provided herein may be administered asthe only therapeutic agent or in combination with other activeingredients. For example, the compounds and compositions may beadministered in combination with other compounds to treat amyloidosesand amyloid disorders, including but not limited to compounds that bindto and stabilize TTR and/or compounds that target TTR RNA and/orcompounds that modulate the expression of the TTR protein and/orcompounds that modulate the transcription of the TTR gene and/orcompounds, which may modulate intra or extracellular protein homeostasisand/or protein stability and/or protein aggregation and/or proteinfolding.

Further active ingredients for combination therapy include but are notrestricted to compounds for the disease modifying or symptomatictreatment of amyloidoses, including but not limited to familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, cerebral amyloidoses,leptomeningeal amyloidis, oculoleptomengial amyloidosis, senile systemicamyloidosis, vitreous amyloidosis, ocular amyloidoses, gastrointestinalamyloidoses, neuropathic amyloidoses, non-neuropathic amyloidoses,nephropathy, non-hereditary amyloidoses, reactive/secondary amyloidoes,Alzheimer's disease, spongiform encephalopathy (i.e. Creutzfeldt Jakobdisease, GSS, fatal familial insomnia), Guillain-Barre' syndrome,frontotemporal dementia, multiple sclerosis, polyneuropathy, Parkinson'sdisease, amyotrophic lateral sclerosis (ALS), Down Syndrome, maculardegeneration, vitreous opacities, glaucoma, type II diabetes andmedullary carcinoma of the thyroid.

Kits

Kits with unit doses of the subject compounds, usually in oral orinjectable doses, are provided. In such kits, in addition to thecontainers containing the unit doses will be an informational packageinsert describing the use and attendant benefits of the drugs intreating pathological condition of interest. Preferred compounds andunit doses are those described herein above.

Articles of Manufacture

The compounds or pharmaceutically acceptable derivatives may be packagedas articles of manufacture containing packaging material, a compound orpharmaceutically acceptable derivative thereof provided herein, which iseffective for modulating TTR folding, or for treatment, prevention oramelioration of one or more symptoms of TTR mediated diseases ordisorders, or diseases or disorders in which TTR misfolding, isimplicated, within the packaging material, and a label that indicatesthat the compound or composition, or pharmaceutically acceptablederivative thereof, is used for modulating TTR folding, or fortreatment, prevention or amelioration of one or more symptoms of TTRmediated diseases or disorders, or diseases or disorders in which TTRmisfolding is implicated.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials,containers, syringes, bottles, and any packaging material suitable for aselected formulation and intended mode of administration and treatment.A wide array of formulations of the compounds and compositions providedherein are contemplated as are a variety of treatments for any diseaseor disorder in which TTR misfolding is implicated as a mediator orcontributor to the symptoms or cause.

Methods

There are a number of published and well established in vitro assaysthat are used to evaluate the ability of test compounds to stabilize theTTR tetramers or prevent TTR fibrils formation. Examples include, butare not limited to, fluorescence polarization assay, fluorescent probebinding assay, isothermal titration calorimetry assay, fibril formationassay, determination of the three-dimensional structure of ligand boundto TTR using X-ray crystallography, FRET, kinetics of transthyretintetramer dissociation or fibril formations, immunoblots to evaluate thestabilizing effect and selectivity of compound binding to TTR in serum.

Provided herein are methods for using the disclosed compounds toincrease the stability of TTR thereby preventing it from misfolding,aggregating and forming TTR amyloid.

The TTR stabilizers disclosed herein may be used to decrease TTR amyloidformation and/or to decrease cell dysfunction and/or death associatedwith TTR amyloid formation. The TTR stabilizers may be used to decreaseTTR amyloid formation in vitro in a cell-free system, in vitro- intra orextracellularily in cell culture, and in vivo, such as TTR found inbodily fluids including but not restricted to blood, serum,cerebrospinal fluid, tissue and organs including but not restricted tothe heart, the kidney, peripheral nerves, meninges, the central nervoussystem, the eye (including the retina and vitreous fluid) of a subject.As such, methods for using the disclosed compounds include administeringthe disclosed compounds in vitro, ex vivo or to a subject in vivo toincrease the stability of TTR found in bodily fluids including but notrestricted to blood, serum, cerebrospinal fluid, tissues and organsincluding but not restricted to the heart, the kidney, peripheralnerves, meninges, the central nervous system, the eyes.

Amyloid fibril formation may be determined using a turbidity assay invitro in a cell-free system. The turbidity assay can use a wild-type TTRor a mutant of TTR with an increased tendency to form amyloid fibrils.When a wild-type TTR is used TTR amyloidogenesis may be initiated byacidification of TTR or the addition of urea. When a mutant of TTR withan increased tendency to form amyloid fibrils, acidification of TTR oraddition of urea may also be used.

TTR stabilizers disclosed herein may be used to decrease TTR amyloidformation in bodily fluids including but not restricted to blood, serum,cerebrospinal fluid, tissue and organs including but not restricted tothe heart, the kidney, peripheral nerves, meninges, the central nervoussystem, the eye (including the retina and vitreous fluid) of a subject.

Also provided are methods for the stabilization of tetramerictransthyretin in a tissue or in a biological fluid, and therebyinhibiting and/or reducing dissociation and/or misfolding of TTRmonomers. Generally, the method comprises administering to the tissue orbiological fluid a composition comprising a stabilizing amount of acompound described herein that binds to transthyretin and preventsdissociation of the transthyretin tetramer by kinetic stabilization ofthe native state of the transthyretin tetramer. As such, methods forusing the disclosed compounds include administering to the tissue orbiological fluid a composition comprising a stabilizing amount of acompound described herein that binds to transthyretin and preventsdissociation of the transthyretin tetramer by kinetic stabilization ofthe native state of the transthyretin tetramer of TTR found in theblood, serum, cerebrospinal fluid, fluids of the central nervous system,retina and the eyes. Generally, the method involves administering to thetissue or biological fluid a stabilizing amount of a compound providedherein that binds to TTR and prevents dissociation of the TTR tetramerby kinetic stabilization of the native state of the TTR tetramer.

Thus, methods which stabilize TTR in a diseased tissue amelioratemisfolding and lessen symptoms of an associated disease and, dependingupon the disease, can contribute to slower progression and/or cure ofthe disease. The extent of misfolding, and therefore the extent ofinhibition achieved by the present methods, can be evaluated by avariety of methods, such as are described in the Examples and ininternational patent application publication no. WO2004/056315. Thedisclosure of the above-referenced application is incorporated herein byreference in its entirety.

Thus, methods, which stabilize transthyretin in a tissue, bodily fluidor organ ameliorate misfolding and lessen symptoms of an associateddisease and, depending upon the disease, can contribute to slowerprogression and/or cure of the disease. The target disease of methods ofthe present disclosure may vary and may include those diseases whichresult from protein misfolding (e.g., TTR folding) or diseasesassociated with an increased tendency to form amyloid fibrils of the TTRtetramer found in the bodily fluids such as blood, serum, cerebrospinalfluid, fluids of the central nervous system and eyes. The extent ofmisfolding, and therefore the extent of inhibition achieved by thepresent methods, can be evaluated by a variety of methods, such as aredescribed in the Examples.

Accordingly, in another aspect the invention includes a method oftreating a TTR amyloid disease, the method comprising administering to asubject diagnosed as having a TTR amyloid disease a therapeuticallyeffective amount of a compound that stabilizes the native state of theTTR tetramer.

In one embodiment, the invention features a method of treating a TTRamyloid disease, the method comprising administering to a subjectdiagnosed as having a TTR amyloid disease a therapeutically effectiveamount of a compound disclosed above that stabilizes TTR tetramer.

The TTR amyloid disease can be, for example, familial amyloidpolyneuropathy, familial amyloid cardiomyopathy, senile systemicamyloidosis, central amyloidosis or ocular amyloidosis.

The subject treated in the present methods can be a human subject,although it is to be understood that the principles of the inventionindicate that the invention is effective with respect to all mammals.

Evaluation of the Activity of the Compounds

A number of in vitro tests can be used to evaluate the compounds fortheir ability to stabilize TTR tetramers or prevent formation offibrils. The tests can include a fibril formation assay, a plasmaselectivity assay, determination of the three-dimensional structure of aTTR: compound complex (e.g., by X-ray crystallography), kinetics of TTRtetramer dissociation or fibril formations, and determining thestoichiometry and energetics of TTR: compound interactions, by, forexample, centrifugation or calorimetry.

The TTR used in the screening methods can be wild type TTR or a mutantTTR, such as a naturally occurring mutant TTR causally associated withthe incidence of a TTR amyloid disease such as familial amyloidpolyneuropathy or familial amyloid cardiomyopathy. Example naturallyoccurring mutant TTRs include, but are not limited to, V122I, V30M, L55P(the mutant nomenclature describes the substitution at a recited aminoacid position, relative to the wild type; see, e.g., Saraiva et al. Hum.Mut. 17:493-503 (2001)).

Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); Room Temperature, RT,rt, and the like.

Materials and Methods

Reagents and Instruments. Prealbumin from human plasma (human TTR) waspurchased from Sigma. Diflunisal, Thyroxine (T4), and resveratrol werepurchased from Fisher. All reactions were carried out under argonatmosphere using dry solvents under anhydrous conditions, unlessotherwise noted. The solvents used were ACS grade from Fisher. Reagentswere purchased from Aldrich and Acros, and used without furtherpurification. Reactions were monitored by thin-layer chromatography(TLC) carried out on 0.20 mm POLYGRAM® SIL silica gel plates (Art.-Nr.805 023) with fluorescent indicator UV254 using UV light as avisualizing agent. Normal phase flash column chromatography was carriedout using Davisil® silica gel (100-200 mesh, Fisher). Wild type TTRconcentration is serum was measured at Stanford Medical School usingnephelometric analyzer (28 mg/dL or 5 μM).

Chemical Synthesis

Methyl 3-(3-bromopropoxy)-4-fluorobenzoate (Compound 2); To a solutionof methyl 4-fluoro-3-hydroxybenzoate 1 (3.0 g, 17.6 mmol, 1 equiv) and1,3-dibromopropane (9.0 ml, 88.2 mmol, 5 equiv) in DMF (40 ml) was addedK₂CO₃ (2.93 g, 21.2 mmol, 1.2 equiv). The reaction mixture was stirredat room temperature for 16 hours. The mixture was diluted with EtOAc(1.5 L), washed with brine (3×0.5 L) and dried with Na₂SO₄. The solutionwas filtered and concentrated. The residue was purified by flash columnchromatography (silica gel, 1-10% EtOAc/hexanes) to afford compound 2(4.21 g, 82% yield); ¹H NMR (CD30D, 600 MHz) δ 7.67-7.61 (m, 2H),7.14-7.07 (m, 1H), 4.21 (t, 2H, J=5.89 Hz), 3.89 (s, 3H), 3.62 (t, 2H,J=6.38 Hz), 2.38-2.31 (m, 2H); (ESI⁺) m/z: calcd for C₁₁H₁₂BrFO₃+H⁺290.00; found 290.01 (M+H⁺).

Methyl 3-(3-(3,5-dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoate(Compound 4); A solution of 2 (780 mg, 2.69 mmol, 1 equiv) in benzene (3ml) was added dropwise to a solution of acetyl acetone (0.552 ml, 5.38mmol, 2 equiv) and DBU (0.804 ml, 5.38 mmol, 2 equiv) in benzene (7 ml).The reaction mixture was stirred at room temperature for 3 days. Themixture was filtered and concentrated. The residue was purified by flashcolumn chromatography (silica gel, 1-10% EtOAc/hexanes) to affordcompound 3 which was used in the next step directly. Hydrazine hydrate(0.36 ml, 6.73 mmol, 2.5 equiv) was added to a solution 3 in ethanol (5ml) and the reaction was heated under reflux for 4 hours. The reactionwas concentrated and purified by flash column chromatography (silicagel, 1-20% MeOH/CH₂Cl₂) to afford compound 4 (288 mg, 35% yield) in twosteps; ¹H NMR (CD₃OD, 600 MHz) δ 7.64-7.58 (m, 2H), 7.20-7.15 (m, 1H),4.01 (t, 2H, J=6.0 Hz), 3.86 (s, 3H), 2.58 (t, 2H, J=7.2 Hz), 2.12 (s,6H), 1.97-1.92 (m, 2H); HRMS (DART) m/z: calcd for C16H19FN2O+H⁺307.1458; found 307.1452 (M+H⁺).

3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzoic acid(Compound VIIc); To a suspension of 4 (100 mg, 0.33 mmol, 1 equiv) in amixture of THF (3 ml) and water (3 ml) was added LiOH·H₂O (27.5 mg, 0.66mmol, 2 equiv). The reaction mixture was stirred at room temperature for14 hr after which it was cooled to 0° C. and carefully acidified to pH2-3 with IN aqueous HCl. The mixture was extracted with EtOAc (3×30 ml)and the combined organic extracts were dried over anhydrous sodiumsulfate and concentrated in vacuo. The crude product was subjected toflash column chromatography (silica gel, 10-50% MeOH/CH₂Cl₂) to giveCompound VIIc (68 mg, 71% yield) as a white solid (>98% purity by HPLC);¹H NMR (CD₃OD, 600 MHz) δ 7.65-7.58 (m, 2H), 7.20-7.14 (m, 1H), 4.00 (t,2H, J=6.0 Hz), 2.58 (t, 2H, J=5.8 Hz), 2.12 (s, 6H), 1.97-1.92 (m, 2H);HRMS (DART) m/z: calcd for C15H17FN2O3+H⁺ 293.1301; found 293.1293(M+H⁺).

3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzamide. To asuspension of 4 (100 mg, 0.33 mmol, 1 equiv) in a mixture of THF (3 ml)and water (3 ml) is added (23.1 mg, 0.66 mmol, 2 equiv) of NH₄OH. Thereaction mixture is stirred at room temperature for 14 hr after which itis cooled to 0° C. and carefully adjusted to pH 7 with IN aqueous HCl.The mixture is extracted with EtOAc (3×30 ml) and the combined organicextracts are dried over anhydrous sodium sulfate and concentrated invacuo. The crude product is subjected to flash column chromatography(silica gel, 10-50% MeOH/CH₂Cl₂) to give3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzamide.

N-ethyl 3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzamide.To a suspension of 4 (100 mg, 0.33 mmol, 1 equiv) in a mixture of THF (3ml) and water (3 ml) is added (27.1 mg, 0.66 mmol, 2 equiv) of C₂H₅NH₂.The reaction mixture is adjusted to pH 9.0 with 0.5N NaOH, then stirredat room temperature for 14 hr after which it is cooled to 0° C. andcarefully adjusted to pH 7 with IN aqueous HCl. The mixture is extractedwith EtOAc (3×30 ml) and the combined organic extracts are dried overanhydrous sodium sulfate and concentrated in vacuo. The crude product issubjected to flash column chromatography (silica gel, 10-50%MeOH/CH₂Cl₂) to give N-ethyl3-(3-(3,5-Dimethyl-1H-pyrazol-4-yl)propoxy)-4-fluorobenzamide.

Isothermal Titration Calorimetry (ITC)

Calorimetric titrations were carried out on a VP-ITC calorimeter(MicroCal, Northhampton, MA). A solution of test compound (CompoundVIIc, Compound A, and Tafamidis) (25 μM in PBS pH 7.4, 100 mM KCl, 1 mMEDTA, 8% DMSO) was prepared and titrated into an ITC cell containing 2μM of TTR in an identical buffer. Prior to each titration, all sampleswere degassed for 10 minutes. 37 injections of test compounds (8.0 μLeach) were injected into the ITC cell (at 25° C.) to the point that TTRwas fully saturated with ligand. Integration of the thermogram after thesubtraction of blanks yielded a binding isotherm that fit best to amodel of two interacting sites exhibiting negative cooperativity. Thedata were fit by a nonlinear least squares approach with four adjustableparameters: K_(d1), ΔH1, K_(d2), and ΔH2 using the ITC data analysismodule in MicroCal ORIGIN 5.0 software.

Fluorescence Polarization Binding Assays

Determination of FP probe displacement by TTR ligands. The affinity oftest compounds to TTR was determined by their ability to displace FPprobe form TTR using our recently developed assay (Alhamadsheh et al.Science Translational Medicine (2011)). In a black 384-well plates (E&KScientific, #EK-31076), FP-probe 5 (200 nM) was incubated with TTR (400nM) in assay buffer (PBS pH 7.4, 0.01% Triton-X100, 1% DMSO in 25 μLfinal volumes) at room temperature. Compound VIIc, Compound A, andtafamidis were then added to the wells a single concentration of 10 μM.The samples were allowed to equilibrate by agitation for 30 min at roomtemperature and fluorescence polarization (excitation λ485 nm, emissionλ525 nm, cutoff λ515 nm) measurements were taken using a SpectraMax M5Microplate Reader (Molecular Devices). The data were fit to thefollowing equation [y=(A−D)/(1+(x/C){circumflex over ( )}B)+D] whereA=maximum FP signal, B=slope, C=apparent binding constant (Kapp), andD=minimum FP signal. The apparent binding constant was reported as themean for triplicate experiments and the best data fit was determined byR² value.

Serum TTR Selectivity Assay

The binding affinity and selectivity of the of test compounds to TTR wasdetermined by their ability to compete for covalent probe 6 (shownbelow) binding to TTR in human serum as previously reported (Choi etal., Bioorg Med Chem (2011)). 98 μL of human serum (Sigma-Aldrich) wasmixed with 1 μL of test compounds (1.0 mM stock solution in DMSO, finalconcentration: 10 μM) and 1 μL of covalent probe 6 (0.36 mM stocksolution in DMSO: final concentration: 3.6 μM). The fluorescence changes(λ_(ex)=328 nm and λ_(em)=384 nm) were monitored every 10 min using amicroplate spectrophotometer reader (Molecular Devices SpectraMax M5)for 6 h at 25° C.

Measurement of Serum WT-TTR Tetramer Stability Against Acid Denaturation

All compounds were 10 mM stock solutions in DMSO and diluted accordinglywith DMSO for different assays. 0.5 μl of each compound was added to24.5 μl of human serum (from human male AB plasma, Sigma) to make thefinal concentration of 10 μM. The samples were incubated at 37° C. for 2h, and then 10 μl of the samples were diluted 1:10 with acidificationbuffer (pH 4.0, 100 mM sodium acetate, 100 mM KCl, 1 mM EDTA, 1 mM DTT).The samples for 0 hours were directly cross-linked with glutaraldehyde(final concentration of 2.5%) for 5 min, and then quenched with 10 μl of7% sodium borohydride solution in 0.1 M NaOH; while the samples inacidification buffer were incubated at room temperature for 72 h andthen cross-linked and quenched with the same protocol. All the sampleswere denatured with adding 100 μl SDS gel loading buffer and boiled for5 min. 12.5 μl of each sample was separated in 12% SDS-PAGE gels andanalyzed by immunoblotting using anti-TTR antiserum (DAKO A0002). Thenormalization was done by dividing each value of the TTR tetramer bandintensity by the average of all the values at time 0.

Measurement of Serum V122I-TTR Tetramer Stability Against AcidDenaturation

Subjects: Samples were obtained from two patients with the V122I TTRmutation. (mutation confirmed by sequencing/test: ‘Amyloidosis DNAtiter’): The western blot analysis was performed as described above forwild type TTR. All the value of the TTR tetramer band intensity wasnormalized by the DMSO treated sample at time 0 which was set as 1.

Cytotoxicity Assay

5×10³ cells were seeded on 80 μl growth media (except 2.5×10⁴ Jurkatcells) in each well in 96-well plates and incubate O/N at 37° C. 20 μlof fresh growth media containing each compound were added into each wellto make the final concentration ranging from 1-100 μM. DMSO was used fornormalization. Cell titer was tested every 24 h using CellTiter 96non-radioactive cell proliferation assay kit (Promega, Madison, WI) at560 nm absorbance.

Design and Synthesis of Compound VIIc

Previously we reported the first high-throughput screen (HTS) for TTRligands, which enabled us to identify a variety of potent andstructurally diverse TTR kinetic stabilizers such as Compound A(Alhamadsheh et al. Science Translational Medicine, 2011). The2-fluorophenyl ring of Compound A occupied the outer binding cavity,placing a portion of the aryl ring into the halogen binding pocket (HBP)1 or 1′. With this observation and additional SAR data obtained fromother ligands, we predicted that introducing a carboxylic acid to the2-fluorophenyl ring of Compound A would allow the molecule to makeadditional electrostatic interactions with K15 and 15′ at the peripheryof the pocket. We hypothesized that the formation of both hydrogen bondsand electrostatic interactions with TTR within the T₄ binding site wouldresult in higher binding affinity and better TTR stabilization. A seriesof analogues of Compound A, including Compound VIIc were synthesizedthat probed the optimal position of the carboxylic acid moiety at the2-fluorophenyl ring. In comparison to the clinical candidate tafamidis,we find that Compound VIIc is a highly effective and selectivestabilizer of both WT and V122I mutant TTR. Compound VIIc also preventsthe dissociation of V122I-TTR in serum obtained from FAC patients veryeffectively. Compound VIIc was not toxic on a number of cell lines,compared to Compound A, which displayed toxicity to certain cell linesat higher concentration (>25 μM). Therefore, by incorporating thecarboxylic acid group we developed Compound VIIc, which is more potentand less toxic than the HTS, Compound A. Compound VIIc has surprisingproperties in comparison to compound A. Biochemical and biophysicalassays revealed important insights into the mechanism of how CompoundVIIc is able to bind V122I-TTR with high selectivity and to stabilizethe TTR tetramer.

Characterization of Compound VIIc Binding Energetics to TTR

The binding affinity of Compound VIIc to TTR at physiological pH wasdetermined using our established fluorescence polarization (FP) assay(FIGS. 1 and 2 ). For comparison, we also tested four known potent TTRkinetic stabilizers (tafamidis and diflunisal are in clinical trials forTTR amyloidosis; T₄ is a natural ligand for TTR; and resveratrol, anatural product that has been shown to prevent TTR aggregation in vitroand TTR-induced cytotoxicity in tissue culture) (FIG. 1 ). The FP assayis a competitive assay that allows measurement of ligands binding to TTRbased on their ability to displace a fluorescent probe from the TTRT₄-binding sites (FP-probe 5, above). All test compounds were able tobind to TTR (purified from human plasma) at 10 μM (FIG. 4 ). The top twocompounds, Compound VIIc and tafamidis, were then assayed in amulti-point dose-response FP assay (concentration range between 0.003and 100 μM). The apparent binding constant of Compound VIIc (K_(app)=193nM, R²=0.994) was similar to that of tafamidis (K_(app)=247 nM,R²=0.990) (FIG. 2 ). Many ligands, including tafamidis (K_(d1)=4.4 nMand K_(d2)=280 nM) (FIG. 3C), bind TTR with negative cooperativity. Weused isothermal titration calorimetry (ITC) to determine the bindingconstants of Compound VIIc to TTR and also to evaluate cooperativitybetween the two TTR T₄ sites. ITC measurements showed that the K_(d1) ofcompound VIIc (K_(d1)=4.8 nM) was an order of magnitude lower than theK_(d1) of compound A (K_(d1)=58 nM), indicating a higher affinity ofcompound VIIc for TTR. Analysis of the free energies associated withCompound VIIc binding to TTR shows high binding affinity and thedissociation constants indicate that Compound VIIc binds TTR withnegative cooperativity (K_(d1)=4.8 nM and K_(d2)=314 nM) (FIG. 3A).Despite the similar binding affinities of Compound VIIc and tafamidis(i.e. similar ΔG values; ΔG₁˜−11.4 and ΔG₂˜−8.8), the binding nature ofboth compounds to TTR is very different. While Compound VIIc binding isalmost entirely enthalpically driven (ΔH1=−13.6 kcal/mol and ΔH2=−7.5kcal/mol), tafamidis binding is about 50% entropy and 50% enthalpy(ΔH1=−5.0 kcal/mol and ΔH2=−3.9 kcal/mol).

Compound VIIc Binds with High Selectivity to WT-TTR in Human Serum

To stabilize the TTR tetramer and thus prevent amyloid formation anddevelopment of cardiac infiltrates in FAC and SSA patients, smallmolecules must be able to selectively bind to TTR in the presence ofmore than 4,000 other human serum proteins. We examined the bindingselectivity of Compound VIIc to TTR in human serum by a fluorescentconjugate competition assay using a covalent probe 6. Covalent probe 6binds selectively to TTR in serum and covalently modifies K15, creatinga fluorescent conjugate. Ligands that bind with high selectivity to TTRin serum decrease covalent probe 6 binding to TTR and therefore lowerthe fluorescence. All test compounds (10 μM) were incubated with humanserum (WT-TTR concentration˜5 μM) in the presence of covalent probe 6(3.6 μM) (FIG. 4 ). Interestingly, Compound VIIc, which binds with highaffinity to TTR in buffer was, when compared to tafamidis and Compound A(70.5±1.4% and 59.3±8.7% probe binding, respectively), the mostselective TTR ligand in serum (3.1±2.9% probe binding) (FIG. 4B).

Our experiments show that both the selectivity as well as the efficacyof TTR kinetic stabilizers can be greatly increased when the ligandorientation within the T₄ pocket is optimized. This is evidenced byCompound VIIc outperforming Compound A and tafamidis in stabilizing TTRat stoichiometric concentrations in human serum. Interestingly, despitethe similar binding affinities of Compound VIIc and tafamidis to TTR inbuffer, their selectivity for TTR in serum is different. Binding toserum proteins is an important factor in determining the overallselectivity, toxicity and pharmacokinetics of a drug. In comparison totafamidis and Compound A, Compound VIIc exhibited remarkable selectivityfor binding TTR in human serum in the presence of more than 4,000 otherhuman serum proteins such as albumin (FIGS. 4-6 ). The exceptionalbinding selectivity of Compound VIIc to serum TTR surpasses that ofTTR's natural ligand, T₄ as well as that of tafamidis and Compound A.

Compound VIIc Increases the Stability of Both WT-TTR and V122I-TTR inHuman Serum Against Acid-Mediated Dissociation and Amyloidogenesis

The compounds were tested for their ability to stabilize WT-TTR in humanserum (FIG. 5 ). TTR tetramer dissociation to monomers and subsequentaggregation occur very inefficiently at neutral pH. To measure thestabilizing effect of Compound VIIc, Compound A, and tafamidis towardsTTR we used an acid-mediated tetramer dissociation assay. Test compounds(10 μM) were pre-incubated with human serum (TTR˜5 μM) and the pH waslowered to pH 4.0 to induce aggregation. Aliquots, taken at 0 and 72hours, were treated with glutaraldehyde to cross-link TTR tetramers thatremained intact in the serum sample. SDS-PAGE followed by immunoblotanalysis was used to measure the amount of intact TTR tetramer after 72hours of acid treatment in the presence and absence of test compounds.In clinical trials of tafamidis the mean maximum concentration (C_(max))of tafamidis in the serum of human subjects, following 20 mg daily dose,was estimated to be around 7.4 μM (Tafamidis Meglumine (Vyndagel)assessment report, European Medicines Agency (EMA) (2011) Procedure No.:EMEA/H/C/002294). At 10 μM, tafamidis stabilized about 58%+/−3% of theTTR tetramer in serum. Compound A provided similar stabilizing effect tothat of tafamidis (61.7%+/−2.1% TTR tetramer stabilization at 10 μM). Incontrast, at 10 μM, Compound VIIc was very effective and stabilized themajority (108+/−6%) of serum WT-TTR. The dramatically increasedstabilizing effect of Compound VIIc compared to Compound A and Tafamidiswas unanticipated and surprising. At 10 μM, Compound VIIc was also veryeffective in stabilizing almost all the V122I-TTR mutant in serumsamples form FAC patients (˜100%) (FIG. 6 ). Due to its ability tostabilize WT- and V122I-TTR, we anticipate Compound VIIc to be effectiveagainst both SSA and FAC.

Compound VIIc Shows No Cytotoxicity In Vitro

In vitro cytotoxicity assays showed that has no in vitro cytotoxicitytowards a panel of cell lines (FIG. 7 ).

The effect of both Compound VIIc and tafamidis on the viability andproliferation of four cell lines was studied via the(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)assay (FIG. 7 ). Compounds were added to the culture medium at differentconcentrations (concentration range from 1 to 100 PM) and the cells wereincubated for additional 24 h at 37° C. Compound VIIc showed nocytotoxic effects towards any of the cell lines that were tested, whileCompound A shows cytotoxicity at high concentrations (>25 μM) towardsHep3B and Hela cells (FIG. 7 ). These data indicate that the CompoundVIIc has less cytotoxicity than Compound A and suggest a better safetyprofile of Compound VIIc. Cytotoxicity results tafamidis were similar toCompound VIIc, except towards MCF7 cells, where cytotoxic effects wereobserved at higher concentrations (>50 μM).

Heterobifunctional Compounds for PPI Disruption

Selection of the linker. The linker is selected considering thebiophysical properties of the interacting proteins (Rp: recruitedprotein, and Tp: target protein). Given the bifunctional compoundeffectively acts to form a protein complex, the long-range forces thatgovern protein-protein interaction are considered when selecting anappropriate linker. The effect of the electrostatic interaction may befavorable, unfavourable or small. If the effect is small, the linker isselected to merely be long enough to project the targeting moiety (T)out of the binding pocket. If the interaction between Rp (recruitedprotein) and Tp (target protein) is disfavoured the linker is selectedto be longer. The longer the linker (e.g., a flexible linker), thegreater the area explored by the targeting element. Although this allowsthe targeting element to bind to the protein Tp, the effectiveconcentration of the T decreases as the area explored increases. Theflexibility of the linker also introduces an entropic cost for smallflexible linkers, due to the restriction of conformational states in thefinal bifunctional complex. As the linker increases in length thispenalty is reduced. Introduction of rigid elements into the linkerrestricts the conformational space explored by T, and provided it issufficiently long allows it to project from the protein, with a reducedconformational penalty. Using a series of linker systems, a library ofbifunctional molecules is prepared for a wide variety of interactingproteins.

Linkers of various length, hydrophilicity, and rigidity are used in thepreparation of bifunctional compounds. The linkers are attached toactivated functional groups on the small molecule targeting andrecruitment moieties using any convenient organic coupling reactions(e.g., ester, amide, and ether bond formation reactions).

Selection of Targeting Moieties. Any small molecule ligand of a targetprotein or target protein/receptor pair can be used. In addition,targeting moieties are identified by small molecule microarrays (SMM)screening for binding to the target protein using computationalapproaches. The attachment site for the linker on the TTR ligand isdetermined by the co-crystal structure of the ligand bound to TTR.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1-32. (canceled)
 33. A compound of Formula (Ib) or (Ic)

wherein X_(a), X_(b), and X_(c), are each independently selected fromC(R⁴)(R⁵), O, N—R⁵ or S; wherein R⁴ and R⁵ are independently selectedfrom hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl, substituted aryl, alkoxy, aryloxy,hydroxyl, a heterocyclic group, halogen, nitro, acyl, substituted acyl,carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl,substituted aminoacyl, amino, substituted amino, acylamino, substitutedacylamino, and cyano; R¹ and R², when present, are each independentlyselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, aryl, substituted aryl, alkoxy, aryloxy, hydroxyl, aheterocyclic group, halogen, nitro, acyl, substituted acyl, carboxyl,alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substitutedaminoacyl, amino, substituted amino, acylamino, substituted acylamino,and cyano; ring A is a 4- to 12-membered ring, in certain embodimentsthe 4- to 12-membered ring is an aromatic or heteroaromatic ring; each Yis independently selected from hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, alkoxy, aryloxy, hydroxyl, a heterocyclic group,halogen, nitro, acyl, substituted acyl, carboxyl, alkoxycarbonyl,substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino,substituted amino, acylamino, substituted acylamino, sulfonamide,sulfonyl fluoride, thioester, and cyano; and c is a number from zero to5; or a pharmaceutically acceptable salt, ester, enol ether, enol ester,amide, acetal, ketal, orthoester, hemiacetal, hemiketal, hydrate,solvate or prodrug thereof.
 34. The compound of claim 33, wherein X_(a),X_(b), and X_(c) are each independently C(R⁴)(R⁵).
 35. The compound ofclaim 33, wherein ring A is a phenyl ring.
 36. The compound of claim 33,wherein c is
 2. 37. The compound of claim 33, wherein each Y isindependently selected from hydrogen, alkyl, halogen, and carboxyl. 38.The compound of claim 33, wherein the compound is a compound of formula(Ib), and R¹ and R² are each independently alkyl.
 39. The compound ofclaim 33, wherein the compound is a compound of formula (Ic), and R¹ ishydrogen.
 40. A compound having the formula (VIIIa)

or a pharmaceutically acceptable salt thereof.
 41. A compound having theformula (VIIb)

or a pharmaceutically acceptable salt thereof.
 42. A pharmaceuticalcomposition comprising the compound of claim 33 and a pharmaceuticallyacceptable carrier, a pharmaceutically acceptable diluent, apharmaceutically acceptable excipient or a pharmaceutically acceptableadjuvant.
 43. A method of treating a transthyretin amyloid disease, themethod comprising administering to a subject in need thereof atherapeutically effective amount of a compound of claim
 33. 44. A kitcomprising a unit dose of a compound of claim
 33. 45. A method ofstabilizing transthyretin (TTR) in a tissue or biological fluid, themethod comprising contacting the tissue or biological fluid with acompound of claim
 33. 46. A method of decreasing amyloid fibrilformation in a tissue or biological fluid, the method comprisingcontacting the tissue or biological fluid with a compound of claim 33.